Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Podsumowanie

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Streszczenie

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

Wprowadzenie

Lung cancer is the leading cause of cancer related deaths worldwide¹. Patients with lung cancer eventually succumb from metastasis to distant organs, notably to the central nervous system, liver, adrenal glands, and bones^2,3,4. Thoracic malignancies have been traditionally classified as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC)⁵. NSCLC is the most frequently diagnosed malignancy and can be subdivided into different histological subtypes, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)⁶. Genomic analysis of resected human primary lung cancers has revealed that tumors within a given histotype can also express diverse molecular perturbations, further contributing to their divergent clinical progression and confounding patient prognosis. The remarkable heterogeneity of lung cancers represents a significant challenge to the rational design, pre-clinical testing, and implementation of effective therapeutic strategies. Consequently, there is a need to expand the repertoire of tractable experimental lung cancer models to study the diverse cellular origins, molecular subtypes, and stages of this disease.

Various approaches using animal models have been employed to study lung cancer in vivo, each with their own advantages and disadvantages depending on the biological question(s) of interest. Genetically engineered mouse models (GEMMs) can target specific genetic alterations in a given progenitor cell type, resulting in tumors that progress within an immunocompetent host⁷. While extremely powerful and clinically relevant, the latency, variability, and/or lung tumor morbidity associated with GEMMs can be prohibitive to certain quantitative measurements and the detection of late stage metastasis in distant organs⁸. A complementary approach is the use of allograft models, whereby lung cancer cells, obtained either directly from a mouse tumor or derived first as established cell lines in culture, are re-introduced into syngeneic hosts. Analogously, lung cancer xenografts are established from human cell lines or patient derived tumor samples. Human cell line xenografts or patient derived xenografts (PDXs) are generally maintained in immunocompromised mice and therefore preclude complete immune-surveillance⁹. Despite this drawback, they provide an avenue to propagate limiting amounts of human biospecimens and study fundamental in vivo properties of human cancer cells, which encode for more complex genomic aberrations than GEMM tumors.

One useful property of allografts and xenografts is that they are amenable to traditional limiting cell dilution assays, employed to quantify the frequency of tumor initiating cells (TICs) within a malignant cell population¹⁰. In these experiments, a defined number of cells are injected subcutaneously into the flank of animals and the frequency of TICs can be estimated based on tumor take rate. Subcutaneous tumors however can be more hypoxic¹¹ and may not model key physiological constraints of the lung tumor microenvironment. Intratracheal delivery of epithelial stem or progenitor cells into the lungs of mice is a method to study pulmonary regeneration and airway stem cell biology¹². However, the engraftment rate from this technique can be relatively low, unless the lungs are first subjected to physiological forms of injury, such as viral infection^13,14. Support from inflammatory stromal cells and/or the disruption of the lung basement membrane may improve retention of transplanted cells into relevant stem cell niches in the distal airways¹⁵. Fibrosis inducing agents can also pre-condition the lungs to enhance engraftment of induced pluripotent cells¹⁶ and mesenchymal stem cells¹⁷. Whether similar forms of airway injury can affect the engraftment rate, tumor initiating capacity, and outgrowth of lung cancer cells has yet to be systematically assessed.

In this study, we describe a method to increase the efficiency of orthotopic lung cancer cell engraftment, by pre-conditioning the lungs of mice with injury. LUAD arises in the distal airways with a significant subset of these cancers developing a fibrotic stroma¹⁸ that often correlates with poor prognosis¹⁹. Bleomycin, a natural nonribosomal hybrid peptide-polyketide, has been extensively utilized to induce pulmonary fibrosis in mice²⁰. Airway instillation of bleomycin first promotes epithelial attrition in the alveoli and recruitment of inflammatory cells, including macrophages, neutrophils and monocytes²¹. This is followed by tissue remodeling in the distal airways, basement membrane reorganization^22,23 and extracellular matrix (ECM) deposition²⁴. The effects of a single bleomycin injection are transient, with fibrosis resolving after 30 days in most studies²⁵. Using both allograft and xenograft models, we tested if pre-conditioning the airways of mice with bleomycin could significantly increase the take rate of LUAD cells in the lungs.

Protokół

All experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Yale University.

1. Set Up / Preparation of the Reagents.

1. Bleomycin
   Caution: Based on the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals, bleomycin is classified as a GHS08 health hazard.
   1. Prepare bleomycin in a chemical hood. Resuspend 15 U into 5 mL of sterile phosphate buffered saline (PBS).
   2. Aliquot 100-200 µL of the solution into glass vials and freeze them at -20 °C for future use. Properly label the tubes with the date of resuspension. Use within 6 months from this date.
      NOTE: Each mouse strain has a different sensitivity to bleomycin²⁶. Testing different doses of bleomycin for each mouse strain is recommended. The mouse strains and the corresponding doses of bleomycin used in representative experiments are listed in Table 1.
2. Mice
   1. Purchase mice needed for the experiment and allow mice 7 days to acclimate before injection. Perform infusions in a biosafety hood. Transfer animals housed in a non-BSL2 compliant room to a BSL2 room using standard institutional procedures prior to bleomycin infusion. Inject mice at 6-8 weeks of age.
      Caution: Inject mice following institutional guidelines for hazardous agents, biosafety level 2 (BSL2) and IACUC approval.
      NOTE: Mice purchased for the representative experiments are listed in the Table of Materials. Only male mice have been used.
3. Lung Cancer Cell Lines
   1. Culture desired cell lines in their respective media. Before injections, perform short tandem repeat DNA profiling or genetic testing to ensure proper cell line identification. To facilitate in vivo and ex vivo imaging, infect cell lines with a lentivirus expressing the thymidine kinase, green fluorescent protein (GFP) and luciferase fusion reporter²⁷, and sort reporter positive cells by fluorescence activated cell sorting (FACS) prior to engraftment. Test lines for mycoplasma every 6 months.
      1. Culture H2030 human cancer cell line as recommended by the manufacturer using Roswell Park Memorial Institute medium (RPMI) with 10% Fetal Bovine Serum (FBS), penicillin-streptomycin, and amphotericin B.
      2. Culture 368T1 murine cell line (derived from a Kras^G12D;p53^-/- mouse) using Dulbecco's Modified Eagle's medium (DMEM) with 10% FBS, penicillin-streptomycin, and amphotericin B.
      3. Culture PC9 human cancer cell line using RPMI with 10% FBS, penicillin-streptomycin, and amphotericin B.

2. Bleomycin Treatment

1. Plan to inject mice intratracheally with bleomycin 14 days prior to tumor cell engraftment.
2. Thaw bleomycin stock on ice 2 h prior to injection.
3. Once thawed, dilute bleomycin to the desired working concentration (0.02 U/50 µL or 0.005 U/50 µL), and keep it on ice.
   NOTE: Concentration of bleomycin depends on the mouse strain used for the experiments (Table 1). Titration of bleomycin in mice should be performed to determine optimal dose.
4. Prepare anesthesia by diluting ketamine and xylazine to a final concentration of 10 mg/mL and 1 mg/mL, respectively, Using a 1 mL syringe and 27 G needle, anesthetize each mouse by injecting intraperitoneally ketamine/xylazine solution at 100/10 mg/kg respectively.
5. Monitor the breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.
6. Place 1 mouse at a time on an intubation platform by hanging its front teeth with its back against the platform.
7. Illuminate the upper chest using a fiber-optic illuminator to help with visualization of the trachea. Open the mouth of the mouse and pull the tongue out gently with sterile flat forceps.
8. Use an intravenous catheter without the needle to avoid blocking breathing. Position the catheter over the white light emitted from the opening of the trachea.
9. Insert the catheter into the trachea until the top of the catheter reaches the front teeth. Confirm proper placement of the catheter in the trachea by visualizing the white light shining through the opening of the catheter in the mouth.
10. Using a pipette, dispense 50 µL of bleomycin or vehicle (PBS) directly into the catheter to ensure that the entire volume is inhaled. Perform this step under sterile conditions.
11. If the catheter is correctly inserted, the mouse will immediately inhale the contents of the catheter. Wait a few seconds until the entire volume travels down the catheter. Then remove the catheter from the trachea and dispose in 10% bleach solution.
    NOTE: The average time per mouse of steps 2.7-2.11 is around 5 min.
12. If the mouse is not inhaling the liquid, carefully monitor breathing and adjust the catheter position. If the mouse stops breathing, remove the catheter immediately and allow the mouse to resume breathing normally before re-inserting the catheter.
13. Place the injected mouse on an IACUC approved heating pad on their back on a flat surface for recovery. The whole procedure will take 10 min per mouse. Do not return an animal that has undergone injection to the company of other animals until fully recovered.
14. Place a "Hazardous Chemical in Use" Card on the cage for 24 h.

3. Monitoring Mice Post-Intubation

1. Monitor the mice for respiratory distress immediately after intubation and then every 15 min until mice wake up from anesthesia. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.
2. Inject ketoprofen (5 mg/kg) intraperitoneally to alleviate pain.
3. Examine the mice 24 h post intubation and 3 times a week for any signs of morbidity.
4. Keep a record of the date of procedure, animal identification, type of procedure, type of anesthetic, and post-operative monitoring observations with times.
5. Monitor the mice for weight loss, respiratory distress, behavioral abnormalities, and a body condition score <2 (segmentation of vertebral column evident/dorsal pelvic bones are palpable) bi-weekly following injection of bleomycin.
6. Euthanize mice under respiratory distress or mice that have experienced 15% loss of body mass by CO₂ or by ketamine/xylazine followed by cervical dislocation. Confirm euthanasia by conducting palpation for respiratory activity.

4. Engraftment of Lung Adenocarcinoma Cell Lines.

NOTE: Perform engraftment of cells 14 days after the injection of bleomycin (step 2.1).

1. Allow cells to grow in 75 cm² flasks undisturbed for 3 days prior to the day of injection with corresponding media as stated in step 1.3.
   NOTE: They should be 80% confluent at the day of injection (which corresponds to 2-3 x 10⁶ in each flask depending on the cell line).
2. Wash the cells growing on 75 cm² flasks with 5 mL of PBS at room temperature.
3. Aspirate PBS and add 1.5 mL of trypsin to the cells and incubate the cells at 37 °C until the cells detach (typically 2-5 min).
   NOTE: We recommend checking every 2 min for cell detachment from the plastic by simple visualization of the bottom of the flask or under a light microscope. Do not exceed 5 min of incubation with trypsin.
4. Neutralize trypsin by adding 4 mL of media containing 10% FBS (same media as step 4.1). Collect the cells into a 15 mL tube and centrifuge it at 200 x g for 3 min at room temperature.
5. Aspirate the media and resuspend the cell pellet in 5 mL of PBS to wash cells. Centrifuge the cells at 200 x g for 3 min at room temperature to pellet cells. Repeat this 1 time.
6. Aspirate the PBS and resuspend the pellet in 1.5 mL of PBS per flask.
7. Mix 50 µL of cell mixture with 50 µL of trypan blue and count the cells using a Cell Counter/Hemocytometer chamber²⁸.
8. Prepare cell suspension by diluting the cells in PBS to inject 1 x 10⁵ cells (or other amount indicated) in 50 µL per mouse. Keep the cells on ice prior to injection.
   NOTE: Prepare at least two times more cells for injection to avoid running out of cell suspension.
9. Prepare anesthesia by diluting ketamine and xylazine to a final concentration of 10 mg/mL and 1 mg/mL, respectively, Using a 1 mL syringe and 27 G needle, anesthetize each mouse by injecting intraperitoneally ketamine/xylazine solution at 100/10 mg/kg respectively.
10. Monitor the breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.
11. Place 1 mouse at a time on an intubation platform by hanging its front teeth with its back against the platform.
12. Illuminate the upper chest using a fiber-optic illuminator to help with visualization of the trachea.
13. Resuspend the cells gently using a p200 pipette to make sure they do not form clumps. Open the mouth of the mouse and pull the tongue out gently with sterile flat forceps.
14. Use an intravenous catheter with the needle removed to avoid blocking breathing. Position the catheter over the white light emitted from the opening of the trachea.
15. Insert the catheter into the trachea until the top of the catheter reaches the front teeth. Confirm proper placement of the catheter in the trachea by visualizing the white light shining through the opening of the catheter in the mouth.
16. Using a pipette, dispense 50 µL of cells directly into the catheter to ensure that the entire volume is inhaled. Perform this step under sterile conditions.
    NOTE: If the catheter is correctly inserted, the mouse will immediately inhale the contents of the catheter.
17. Wait a few seconds until the entire volume travels down the catheter, and then remove the catheter from the trachea and dispose in 10% bleach solution.
18. If the mouse is not inhaling the liquid, carefully monitor breathing and adjust the catheter position. If the mouse stops breathing, remove the catheter immediately and allow the mouse to resume breathing normally before re-inserting the catheter.
    NOTE: The average time per mouse of steps 4.11-4.18 is around 5 min.
19. Place the injected mouse on an IACUC approved heating pad on their back on a flat surface for recovery.
    NOTE: The whole procedure will take 10 min per mouse. Do not return an animal that has undergone injection to the company of other animals until fully recovered.
20. Shave the entire rib area both ventrally and dorsally of B6129SF1/J and other mouse strains with dark hair using an electric shaver prior to imaging.
21. 2-3 min after cell injection, inject 100 µL of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Alternate the eye used for injection every imaging session.
22. After at least 2 min, place up to 5 mice in an animal bioluminescence imager in the dorsal recumbency position and acquire a ventral picture using luminescence settings²⁹.
23. Under the control panel adjust the resolution and sensitivity settings to measure luminescence of a given cell line. For most applications, start with 3 min (or "Auto" if saturated), binning=Medium (4), F/Stop=1, Field of View=D, Subject Height=1.50.
    NOTE: If the injection is successful, luminescence signal is detected in the upper chest area, in one lung or both lungs.
24. Flip mice onto sternal recumbency position and acquire a dorsal picture as above.
    NOTE: If cell injection is not performed properly, cells may cluster at the entrance of the trachea or in the neck.
25. Inject ketoprofen 5 mg/kg intraperitoneally to alleviate pain.
26. Move mice back to their cage and place a "BSL-2 Agent in Use" Card on the cage.
27. Monitor mice for respiratory distress immediately after intubation, and then every 15 min until mice wake up from anesthesia. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.
28. Examine the mice 24 h post intubation and three times a week for any signs of morbidity.
29. Keep record in the logbook of the date of procedure, animal identification, type of procedure, type of anesthetic and post-operative monitoring observations and times.
30. Monitor mice for weight loss, respiratory distress, behavioral abnormalities, and a body condition score <2 (segmentation of vertebral column evident/dorsal pelvic bones are palpable) bi-weekly following inoculation of bleomycin.
    NOTE: Mice with lung tumors may exhibit respiratory irritation, weight loss, cachexia, and/or death.
31. Euthanize mice under respiratory distress or mice that have experienced 15% loss of body mass by CO₂ or by ketamine/xylazine followed by cervical dislocation if collecting tissues. Confirm euthanasia by conducting palpation for respiratory activity.

5. Monitoring of Tumor Growth by Bioluminescence Imaging

1. Repeat imaging of the mice at day 3 post-engraftment and weekly thereafter.
2. Anesthetize mice by injecting ketamine/xylazine (as described in steps 2.4-2.5) or by inhaled isoflurane (2%). Monitor breathing of mice and employ a toe pinch to confirm proper anesthetization. Apply vet ointment on eyes to prevent dryness while under anesthesia.
3. Shave the entire rib area both ventrally and dorsally of B6129SF1/J and other mouse strains with dark hair using an electric shaver prior to imaging.
4. Once the mice are under anesthesia, inject 100 µL of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Wait 2 min. Alternate the eye used for injection every imaging session.
5. Place mice in the imager and acquire dorsal and ventral images as described in steps 4.22-4.24.
6. Place mice back into the cage after imaging, on their back on a flat surface for recovery.
   NOTE: The whole procedure will take 5 min per group of 5 mice. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.
7. Analyze the images using the imager/bioluminescence analysis software.
   1. Select the appropriate images and load them as a group in the bioluminescence analysis software.
   2. Click ROI tool | square to add a square region of interest (ROI). Place the ROI over the upper chest area, covering the entire lung image. Repeat this for each mouse image.
   3. Right click and select Copy all ROI function to apply the same ROI to all images in the group and position them in the upper chest of the mice, both ventral and dorsal views.
   4. In the upper left corner of the image window, select the Photons function. Click the Measure control panel function to measure the ROIs. Copy and paste the output table containing total flux (photons/s) into a software of choice to analyze the data further.
   5. Normalize the daily ROI value of each mouse to its ROI value measured on day 0.

6. Tissue Isolation and Processing.

1. Anesthetize mice by injecting ketamine/xylazine (as described in steps 2.4-2.5 or by inhaled isoflurane (2%). Monitor breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.
2. Inject 100 µL of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Wait 2 min.
3. Image mice following steps 4.22-4.24 from this protocol.
4. Inject into the other eye from step 6.2 another 100 µL of luciferin retro-orbitally using an insulin needle prior to euthanasia.
5. Euthanize the mice by intraperitoneal injection of ketamine/xylazine (see step 2.4 for details) followed by cervical dislocation if mice are under deep anesthesia in accordance with guidelines provided by IACUC. Confirm euthanasia by conducting palpation for respiratory activity.
6. Using sterile surgical scissors make a skin incision below the sternum. Open the muscle layer along the diaphragm using forceps and surgical scissors and expose the thoracic cavity by cutting through the rib cage on either of the lateral sides avoiding the internal thoracic arteries.
7. Perfuse the mouse by making a small incision in the right atrium of the heart and inject 5 mL of PBS through the left ventricle. The color of lung tissue will go from red to pale pink-white if the blood perfusion is done properly.
8. Excise the lungs from the thoracic cavity carefully by grabbing the esophagus or heart with forceps. Gently pull the tissue and use surgical scissors to carefully cut connecting tissue avoiding puncture of the lung tissue. Preserve the trachea as much as possible for inflation of the lung airways at a later step.
9. Wash the lung by submerging and swirling the tissue 3 times in a 6 well plate filled with 3 mL of PBS to remove excess blood.
10. Place the lung onto the lid of a 6 well plate, and place the lid containing the tissue in the bioluminescent imager and acquire a luminescent image³⁰.
    NOTE: We recommend selecting the following settings from the "Acquisition control panel" "Field of view=A" and "Subject Height=0.75 cm" and then "Auto exposure" to avoid getting saturated images.
11. Excise and image other organs, as done in step 6.9-6.10, where metastasis have been identified by luminescence, such as brain, liver, adrenal, bone, lymph nodes, kidney or spleen³¹.
12. Perfuse the lung with 1 mL of 4% paraformaldehyde by inserting the needle into the trachea of the mouse. The lungs will inflate if well-perfused.
    Caution: 4% paraformaldehyde is a toxic, a health hazard GHS07 and GHS08.
13. Transfer the lungs into a 15 mL conical with 5 mL of 4% paraformaldehyde.
14. Fix the lungs or other tissues by incubating the tissue with 4% paraformaldehyde overnight at 4 °C in a shaking device at 30-50 rpm.
15. Embed lungs (or other tissues) in paraffin or optimum cutting temperature compound depending on the application³².
    NOTE: Paraffin is the preferred method to preserve the lung structure and for Masson Trichrome and Hematoxylin-Eosin staining.

Wyniki

To increase the efficiency of LUAD cancer cell engraftment into the lungs of mice, we developed a protocol that first pre-conditions the airways using bleomycin followed by orthotopic tumor cell injection (Figure 1). We confirmed that even when administered into immunocompromised athymic mice, bleomycin induced transient fibrosis by day 14 as evidenced by loss of airway architecture and increased collagen deposition (Figure 2). G...

Dyskusje

Striking clinical parallels have been documented between lung cancer and other chronic diseases of the lung³⁶. In particular, patients with idiopathic pulmonary fibrosis (IPF) have an increased predilection for developing lung cancer, and this association is independent of smoking history^37,38. IPF is characterized by progressive destruction of lung architecture and impaired respiratory function through deposition of ECM^3...

Materiały

Name	Company	Catalog Number	Comments
Bleomycin	Sigma	B5507-15UN	CAUTION Health hazard GHS08
Exel Catheter 24G	Fisher	1484121	Remove needle. For intratracheal injection
Ketamine (Ketaset inl 100 mg/mL C3N 10 mL)	Butler Schein	56344	To anesthetize mice
Xylazine	Butler Schein	33198	To anesthetize mice
Ketoprofen, 5,000 mg	Cayman Chemical	10006661	Analgesic
Puralube Veterinary Ophthalmic Ointment	BUTLER ANIMAL HEALTH COMPANY LLC	8897	To prevent eye dryness while under anesthesia
D-Luciferin powder	Perkin Elmer Health Sciences Inc	122799	For luminescent imaging. Reconstitute powder with PBS for a working concentration of 15mg/mL. Protect from Light
Rodent Intubation stand	Braintree Scientific	RIS-100	Recommended stand for intratracheal injection
MI-150 ILLUMINATOR 150W MI-150	DOLAN-JENNER INDUSTRIES	MI-150 / EEG2823M	To illuminate and visualize trachea
Graefe Forceps, 2.75 (7 cm) long serrat	Roboz	RS-5111	For intratracheal injection
Syringe Luer-Lok Sterile 5ml	BD / Fisher	309646
Satiny Smooth by Conair Dual Foil Wet/Dry Rechargeable Shaver	Conair	-	To shave mice
Bonn Scissors, 3.5" straight 15 mm sharp/sharp sure cut blades	Roboz	RS-5840SC
15 mL conical tube	BD / Fisher	352097
1.5 mL centrifuge tubes	USA SCIENTIFIC INC	1615-5500
Vial Scintillation 7 mL Borosilicate Glass GPI	Fisher	701350
Filter pipette tips (200 μL)	USA SCIENTIFIC INC	1120-8710
Phosphate Buffered Saline	Life Technologies	14190-144
0.25% Trypsin-EDTA	Life Technologies	25200-056
DMEM high glucose	Life Technologies	11965-092
RPMI Medium 1640	Life Technologies	11875-093
Fetal bovine serum USDA	Life Technologies	10437-028
Penicillin-Streptomycin	Life Technologies	15140-122
Amphotericin B	Sigma	A2942-20ML
Trypan Blue Stain 0.4%	Life Technologies	15250-061
Countess Automated Cell Counter	Life Technologies	AMQAX1000
Flask T/C 75cm sq canted neck, blue cap	Fisher / Corning	353135
IVIS Spectrum Xenogen Bioluminiscence	Perkin Elmer Health Sciences Inc	124262	For in vivo bioluminescence imaging
Living image software	Perkin Elmer Health Sciences Inc	128113	For in vivo bioluminescence analysis
XGI-8 Gas Anesthesia System	Perkin Elmer Health Sciences Inc	118918	For Isoflurane anesthesia
BD Ultra-Fine II Short Needle Insulin Syringe 1 cc. 31 G x 8 mm (5/16 in)	BD / Fisher	BD328418	For retro-orbital luciferin injection
Syringe 1ml	BD / Fisher	14-823-434	For intraperitoneal injections
26 G x 1/2 in. needle	BD / Fisher	305111	For intraperitoneal injections
4% Paraformaldehyde	VWR	43368-9M	CAUTION Health hazard GHS07, GHS08. For fixing tissue
Pipet-Lite Pipette, Unv. SL-200XLS+	METTLER-TOLEDO INTERNATIONAL	17014411
Mayer's Hematoxylin	ELECTRON MICROSCOPY SCIENCES	517-28-2
Eosin Y stain 0.25% (w/v) in 57%	Fisher	67-63-0
Masson Trichrome Stain Kit	IMEB Inc	K7228	For masson trichrome stain to visualize collagen
Superfrost plus glass slides	Fisher	1255015
6 well plate	Corning	C3516
Universal Mycoplasma Detection Kit	ATCC	30-1012K
OCT Embedding compound	ELECTRON MICROSCOPY SCIENCES	62550-12	For embedding tissue for frozen sections
Leica CM3050 S Research Cryostat	Leica	CM3050 S	To section tissue for staining analysis
Keyence All-in One Fluorescence Microscope	Keyence	BZ-X700
ImageJ	US National Institutes of Health	IJ1.46	http://rsbweb.nih.gov/ij/ download.html
Prism 7.0 for Mac OS X	GraphPad Software, Inc.	-
Athymic (Crl:NU(NCr)-Foxn1nu) mice	Charles River	NIH-553
NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice	Jackson Laboratories	5557
B6129SF1/J mice	Jackson Laboratories	101043
NIH-H2030 cells	ATCC	CRL-5914
368T1	generously provided by Monte Winslow (Standford University)	-
PC9 cells	Nguyen DX et al. Cell. 2009;138:51–62	-
H2030 BrM3 cells	Nguyen DX et al. Cell. 2009;138:51–62	-